The Section on Molecular Neuroscience studies the molecular mechanisms of chemically coded ionotropic and metabotropic neurotransmission in the nervous system. The ultimate goals of the project are identifying molecular components of synaptic transmission, and how these components are regulated to allow short-term and long-term information to be encoded within postsynaptic neurons and neuroendocrine cells. Advances have been made in the following areas in our laboratory: 1. We have demonstrated, with our collaborators in NICHHD and NINDS, that chromogranin A is a 'master switch' controlling the formation of the large dense core secretory granules from which endocrine hormones and neuropeptides are released during neurotransmission. When chromogranin A is expressed in non-endocrine cells, large dense-core granules are made, and when chromogranin A expression is knocked-down in neuroendocrine cells, granules disappear (as do co-stored secretory proteins, but not their mRNAs), but re-appear (as do co-stored secretory proteins) if chromogranin A is re-introduced (T. Kim, J.-H.Tao-Cheng, L. E. Eiden and Y. P. Loh, Chromogranin A, an ?On/Off? switch controlling dense-core secretory granule biogenesis, Cell 106: 499-509, 2001). This work reveals the primordial role of chromogranin A in initiating a cascade of cellular events leading to sequestration of neuropeptides and hormones in large dense-core vesicles, and their secretion from these vesicles. We will now study the physiological consequences of selective and conditional ablation of large dense core granule-mediated neurotransmission in several nervous and endocrine systems, and in several cell culture models for regulated neurosecretion, by manipulation of this 'master switch'. 2. We have developed a mouse deficient in one of the neuropeptides stored in large dense-core granules to study its role in synaptic transmission of information in the autonomic nervous system. Loss of pituitary adenylate cyclase activating peptide (PACAP) from the preganglionic inputs to the adrenal medulla seems to affect the ability of this synapse to response to long-term metabolic stress by up-regulating catecholamine biosynthetic enzymes. The PACAP-deficient mouse fails to respond to prolonged hypoglycemia induced by elevated insulin levels by sustained release of the gluconeogenic hormone epinephrine from the adrenal gland, due to a failure to sustain epinephrine biosynthesis via induction of its biosynthetic enzyme tyrosine hydroxylase. PACAP-deficient mice are thus susceptible to insulin-induced hypoglycemia while normal mice are not. This is the first demonstration of a neuropeptide?s role in trans-synaptic induction of the catecholamine biosynthetic enzyme tyrosine hydroxylase. We take it as a clue to the evolutionary importance of neuropeptide co-transmission at classical neurotransmitter synapses, that of providing an emergency or stress response capability to the synapse for responding to long-term stress by increasing the capacity for classical hormone production and secretion (C. Hamelink, O.Tjurmina, R. Damadzic, W. S. Young, E. Weihe, H.-W. Lee and L. E. Eiden, PACAP is a sympathoadrenal neurotransmitter involved in catecholammine regulation and glucohomeostasis, Proc. Natl. Acad. Sci. USA, submitted for publication and in review). 3. We discovered last year a combinatorial signaling system activated by the neuropeptide PACAP that targets the VIP gene in neuroendocrine cells through two novel pathways involving calcineurin and cAMP-dependent/protein kinase A-independent signaling, respectively, and are in the process of constructing PC12 cell lines with forced expression of dominant negative putative components of this new signaling pathway to characterize it in full and determine its role in the nervous system in vivo via examination of the onset of expression of the relevant signaling components in the developing nervous system, and biochemical complementation in our PACAP knock-out mouse model.